piggyBac转座子介导HepG2细胞不同亚型药物代谢酶稳定共表达方法比较  

Comparison of methods for stable co-expression of different subtype drug-matabolizing enzymes in HepG2 cells by piggyBac transposon

作　　者：庞士慧 钟国瑞 谢水林[1] 李浩健 邹淑香 贾英[1] 戴仁科[1] 黄黎珍[1,2] PANG Shi-hui;ZHONG Guo-rui;XIE Shui-lin;LI Hao-jian;ZOU Shu-xiang;JIA Ying;DAI Ren-ke;HUANG Li-zhen(School of Bioscience and Bioengineering;Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou 510006, China)

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006 [2]华南理工大学发酵和酶工程重点实验室,广东广州510006

出　　处：《中国药理学与毒理学杂志》2018年第2期125-134,共10页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金项目(81202585);广东省科技计划项目(2014A030304014)~~

摘　　要：目的在piggy Bac(PB)转座子介导下,探索在HepG2细胞中实现多个药物代谢酶稳定共表达的策略,为建立体外药物代谢和肝毒性研究的理想细胞模型奠定基础。方法首先选择3种目前针对大片段DNA使用较广的转染方法:Lipofectamine~?LTX,Gen Jet^(TM)(Ver.Ⅱ)和Neon~?Transfection System,分别转染N端标记增强型绿色荧光蛋白质粒(pEGFP-N2)和2A串联重组细胞色素3A4(CYP3A4)和CYP2C19的PB转座子质粒(pPB-CYP3A4-2A-2C19)至HepG2细胞,48 h后比较不同方法的转染效率和细胞毒性,确立高效低毒的HepG2细胞转染方法。然后选择该法进行转染,将设计的3组PB重组转座子:单基因转座子(PB-CYP3A4)、2A串联多基因转座子(PB-CYP3A4-2A-2C19)、多个单基因转座子混合物[PB-CYP3A4,PB-CYP2C8,PB-CYP2A6,有机阴离子转运多肽1B1的PB转座子(PB-OATP1B1)]分别转染HepG2细胞,利用嘌呤霉素和GFP进行单克隆细胞筛选:挑选具有抗性且表达GFP的细胞单克隆并进行扩大培养,采用实时荧光定量PCR、Western蛋白质印迹和高效液相色谱-串联质谱联用法分别检测各组药物代谢酶m RNA、蛋白质水平及酶活性,并进行统计分析。结果 3种转染方法比较发现,Gen Jet^(TM)法的转染效率显著高于其他2种(P<0.01),介导pEGFP-N2和pPB-CYP3A4-2A-2C19的转染效率分别高达(94.2±2.5)%和(89.3±3.3)%,且该法具有较低的细胞毒性。因此选择Gen Jet^(TM)法进行后续PB转座子转染。分别转染3组PB重组转座子发现,单个转座子PB-CYP3A4和PB-CYP3A4-2A-2C19转染后,筛选获得的抗性细胞克隆中各个药物代谢酶在m RNA、蛋白质及活性水平均显著提高,其中2A可实现CYP3A4和CYP2C19药物代谢酶协同稳定表达;而多个转座子混合共转染细胞中,仅有随机几个基因表达;且各药物代谢酶基因的表达不均衡,仅CYP3A4药物代谢酶基因在单克隆细胞中表达水平明显升高。结论 Gen Jet^(TM)法可成为PB重组转座子转染HepG2细胞的有效方法。OBJECTIVE To study the methodology of achieving stable co-expression of drug-metabolizing enzymes in the HepG2 cells by the piggy Bac（PB） transposon system. METHODS N-terminal attachment of enhanced green fluorscent protein plasmid（pEGFP-N2） and 2 A peptide linked recombinant PB transposon plasmid containing dual-genes encoding drug metabolizing enzymes cytochrome P450 3 A4（CYP3A4） and CYP2 C19（pPB-CYP3A4-2 A-2 C19） were transfected into HepG2 cells respectively by LipofectamineLTX reagent, GenJet（TM）（Ver.Ⅱ） reagent and NeonTransfection System reagent, which were widely used for large-sized DNA fragments transfection. 48 h later, the transfection efficiency and cell toxicity were detected and compared between the three methods so as to find a method with relatively high efficiency and low toxicity for later transfection. Then, three groups of recombinant PB transposons-single-gene transposon（PB-CYP3A4）, 2 A peptide linked dual-gene transposon（PB-CYP3A4-2 A-2 C19） and multiple single-gene transposon mixture[PB-CYP3A4, PBCYP2 C8, PB-CYP2 A6, organic anion transporting polypeptide 1 B1 PB transposon（PB-OATP1 B1）]-were transfected into HepG2 cells respectively with the above established method. The puromycin（Puro）-resistant and GFP positive cell clones were picked up and further cultured. The mRNA, protein and metabolic levels of drug-metabolizing enzymes in monoclonal cell lines were detected by quantitative real-time PCR, Western blotting and high performance liquid chromatography-tandem mass spectrometry respectively after screening by Puro and green fluorescence. Comparisons of different groups were made using statistical analysis. RESULTS The comparison of three different transfection methods indicated that the transfection efficiency of GenJet^TM was up to（94.2±2.5）% and（89.3±3.3）%, significantly higher than those of the other two methods（P0.01）, along with lower cytotoxicity. Then GenJet^TM was chosen for later transfection. In the Puro-resistant monoclon

关 键 词：PIGGYBAC转座子 HEPG2细胞 药物代谢酶 多基因共表达 

分 类 号：R969.1[医药卫生—药理学]